Articular cartilage is a specialized tissue found at the end of articulating bones. Cartilage, unlike other connective tissues, lacks blood vessels, nerves, lymphatics and basement membrane. It is responsible for the distribution of load resistance to compressive forces, and the smooth gliding that is part of joint function.
Cartilage is composed of chondrocytes which synthesize an abundant extracellular matrix, which is composed of water, collagens, proteoglycans and noncollagenous proteins and lipids. Collagen serves to trap proteoglycans and to provide tensile strength to the tissue. Type II collagen is the predominant collagen in cartilage tissue. The proteoglycans are composed of a variable number of glycosaminoglycan chains, keratan sulphate, chondroitin sulphate and/or dermatan sulphate, and N-linked and O-linked oligosaccharides covalently bound to a protein core. The sulphated glycosaminoglycans are negatively charged resulting in an osmotic swelling pressure that draws in water.
Although, histologically, articular cartilage appears very homogenous, the matrix organization and composition differ from the superficial to the deep zones (Aydelotte and Kuettner, Conn. Tiss. Res. 18: 205, 1988; Zanetti et al, J. Cell Biol. 101: 53, 1985; and Poole et al, J. Anat. 138: 13, 1984). Articular cartilage appears to be composed of zones which show a characteristic gradation of features from the surface of the tissue to the base of the tissue adjacent to the bone. In the superficial zone, for example, chondrocytes are flattened and lie parallel to the surface embedded in a matrix that contains tangentially arranged collagen and few proteoglycans. In the mid zone, chondrocytes are spherical and surrounded by a matrix rich in proteoglycans and obliquely organized collagen fibers. In the deep zone, close to the bone, the collagen fibers are vertically oriented. The keratan sulphate rich proteoglycans increase in concentration with increasing distance from the cartilage surface (Zanetti et al, supra).
Ultrastructural variation is evident between the pericellular and interterritorial matrix areas. The pericellular collagen fibers are much thinner and do not exhibit the usual 68 nm periodicity characteristic of collagen in the interterritorial zone. It is not known how these zones are established or maintained (Poole et al, supra: Urban and Bayliss, Biochem. Biophys. Acta. 992: 59, 1989; Brown et al, Conn. Tiss. Res. 24: 157, 1990; Schneiderman et al J. Orthop. Res. 4: 393, 1986).
Studies of cartilage organization and pathophysiology have been severely restricted by the limited availability of cartilage tissue and the inability of in vitro culture systems to mimic the organization of in vivo cartilage tissue. In vivo cartilage has been removed and maintained in explant cultures, but there are several problems associated with these cultures (Poole et al supra Lane and Brighton, Arth. Rheum 17: 235, 1974; and Morales et al, J. Biol. Chem. 259: 6720, 1984). There is a loss of matrix molecules from the cartilage during culture, and the amount of cartilage available for experimentation is therefore limited. Chondrocytes have been isolated from cartilage and grown in monolayer culture systems (Manning and Bonner, Arth. Rheum. 10: 235, 1967; Horwitz and Dorfman, J. Cell Biol. 45: 434, 1970; and Green, Clin. Orthop. Rel. Res. 75: 248, 1971). However, chondrocyte phenotype in these cultures is labile and the chondrocytes dedifferentiate to fibroblasts, as defined by production of type I collagen and small nonaggregating proteoglycans (Von der Mark et al, Nature 267: 531, 1977; and Solursh, Am. J. Med. Gen. 34: 30, 1989).
Maintaining chondrocytes in a spherical shape has been shown to slow down or prevent dedifferentiation of the chondrocyte phenotype (Watt and Dudhia, Differentiation 140, 1988). Accordingly, culture systems have been developed to maintain the cells in a spherical shape. Primary cell cultures either plated at high density in monolayer or droplet form, in suspension culture, in collagen gel, in or on agarose gel, in composition agarose-collagen gels, in alginate or on apatite beads, or a combination of monolayer culture followed by transfer to agarose culture have been employed in an attempt to slow down or prevent dedifferentiation (Kuettner et al, J. Cell. Biol. 93: 743, 1982; Van Kampen and Veldhuijzen, Exp. Cell. Res. 140: 440, 1982; Delbruck et al, Conn. Tiss. Res. 15: 155, 1986; Thompson et al, Exp. Cell. Res. 157: 483, 1985; Bassler et al, In Vitro 22: 113, 1986; Cheung, In Vitro Cell. Dev. Biol. 21: 353, 1985; Guo et al, Conn. Tiss. Res. 19: 277, 1989; Aulthouse et al, In Vitro 25: 659, 1989; and Solursh, J. Cell. Biochem. 45: 258, 1991).
Alternative approaches to maintaining the chondrocyte phenotype have been to induce chondrogenesis by growing mesenchymal cells in diffusion chambers or in monolayer in the presence of beta TGF (O'Driscoll et al, Trans. Orthop. Res. 37: 125, 1991 and Nakahara et al, Bone 11: 181, 1990), or perichondrial grafts (Amiel et al, Conn. Tiss. Res. 18: 27, 1988). Under all of the above conditions the chondrocytes maintain at least a partial chondrocyte phenotype, as indicated by the synthesis of type II collagen and proteoglycans specific to articular cartilage. However, there are a number of problems with these types of cultures. Proliferation in some of the cultures is inhibited so that only a limited number of cells can be generated. It has also proved difficult to isolate cells from these cultures. The most important limitation, however, is that none of these culture systems can mimic the in vivo morphology of articular cartilage, including the cellular and matrix organization described above.
Green (Clin. Orth. Rel. Res. 75:248. 1971) teaches a process for growing chondrocytes in vitro in pelleted aggregate cultures on Millipore cellulose acetate inserts. Green describes the in vitro production of a chondro-myxoid matrix by rabbit chondrocytes. Kuettner et al (J. Cell. Biol. 93:751, (1982)) describe methods of culturing bovine chondrocytes on plastic dishes. Kuettner in U.S. Pat. No. 4,356,261 describes methods of culturing chondrocytes in suspension culture in roller bottles. Type II collagen was reported as being the major matrix-associated collagen synthesized in vitro. Kuettner et al analyzed proteoglycans synthesized by the chondrocytes in their culture system by chromatography of .sup.35 S pulse-labelled cultures. The proteoglycans synthesized were compared with those of in vivo bovine articular cartilage. Bassler et al (1986) teach a suspension culture of human chondrocytes wherein aggregates of chondrocytes with secreted matrix were produced by a gyratory shaker. Type II collagen and proteoglycans were detected in the secreted matrix by immunofluoresence and radioimmunoassay.
Cheung (In Vitro Cell. Dev. Biol. 21:353, 1985) teaches a method of culturing canine chondrocytes on porous hydroxyapatite ceramic granules. The cells reportedly proliferated and secreted metachromatic extracellular matrix for up to 13 months. An agarose gel matrix has also been described as suitable for the in vitro culture of human chondrocytes (Delbruck et al, Conn. Tiss. Res. 15:155, 1986). Delbruck et al disclosed human chondrocytes distributed in the agarose and forming a pericellular region surrounded by an interterritorial-like region. Type II collagen was detected in the gel matrix cultures by immunofluoresence and acid soluble collagens were examined by SDS polyacrylamide gel electrophoresis.
Watt and Dudhia (Differentiation 38:140, 1988) disclose a composite gel of collagen and agarose for the culture of porcine chondrocytes. The composite gel prevented chondrocytes spreading. However, virtually no extracellular matrix was secreted in the low density culture composite gels.
Macklis et al (In Vitro Cell. Develop. Biol. 21:180, 1985) teach a collagen surface for culturing peripheral nervous system cells, comprising collagen derivatized to polystyrene plastic culture dishes. Macklis et al disclose that the derivatized coating process yielded enhanced collagen adhesion and increased long term survival of cultured nerve cells, compared to collagen coating produced by absorption techniques.